Bioconjugate Chem. 2002, 13, 845−854
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Polyethylenimine-graft-Poly(ethylene glycol) Copolymers: Influence of Copolymer Block Structure on DNA Complexation and Biological Activities as Gene Delivery System Holger Petersen,† Petra M. Fechner,† Alison L. Martin,‡ Klaus Kunath,† Snjezana Stolnik,‡ Clive J. Roberts,‡ Dagmar Fischer,† Martyn C. Davies,‡ and Thomas Kissel*,† Department of Pharmaceutics and Biopharmacy, Philipps University, Ketzerbach 63, D-35032 Marburg, Germany, and School of Pharmaceutical Sciences, University of Nottingham, University Park, Nottingham, NG7 2RD, United Kingdom . Received March 18, 2002; Revised Manuscript Received April 25, 2002
For two series of polyethylenimine-graft-poly(ethylene glycol) (PEI-g-PEG) block copolymers, the influence of copolymer structure on DNA complexation was investigated and physicochemical properties of these complexes were compared with the results of blood compatibility, cytotoxicity, and transfection activity assays. In the first series, PEI (25 kDa) was grafted to different degrees of substitution with PEG (5 kDa) and in the second series the molecular weight (MW) of PEG was varied (550 Da to 20 kDa). Using atomic force microscopy, we found that the copolymer block structure strongly influenced the DNA complex size and morphology: PEG 5 kDa significantly reduced the diameter of the spherical complexes from 142 ( 59 to 61 ( 28 nm. With increasing degree of PEG grafting, complexation of DNA was impeded and complexes lost their spherical shape. Copolymers with PEG 20 kDa yielded small, compact complexes with DNA (51 ( 23 nm) whereas copolymers with PEG 550 Da resulted in large and diffuse structures (130 ( 60 nm). The ζ-potential of complexes was reduced with increasing degree of PEG grafting if MW g 5 kDa. PEG 550 Da did not shield positive charges of PEI sufficiently leading to hemolysis and erythrocyte aggregation. Cytotoxicity (lactate dehydrogenase assay) was independent of MW of PEG but affected by the degree of PEG substitution: all copolymers with more than six PEG blocks formed DNA complexes of low toxicity. Finally, transfection efficiency of the complexes was studied. The combination of large particles, low toxicity, and high positive surface charge as in the case of copolymers with many PEG 550 Da blocks proved to be most efficient for in vitro gene transfer. To conclude, the degree of PEGylation and the MW of PEG were found to strongly influence DNA condensation of PEI and therefore also affect the biological activity of the PEI-g-PEG/ DNA complexes. These results provide a basis for the rational design of block copolymer gene delivery systems.
INTRODUCTION
Surface modification of drug delivery systems with PEG1 has met with increasing interest to enhance biocompatibility (1), increase systemic circulation times (2), and alter their biodistribution (3). For instance, nanoparticles (4), liposomes (5), and also adenoviruses (6) have been “PEGylated” mainly to obtain long-circulating particulate delivery systems, based on the so-called “stealth” effect. Recently, PEGylation methodology has been reviewed (7). PEGylation of polycations for gene delivery has also been realized leading to block or graft copolymers as summarized in refs 8 and 9. Among other polycations, PLL has been modified by PEGylation (10* To whom correspondence should be addressed. Tel: +496421-282-5881. Fax: +49-6421-282-7016. E-mail kissel@ mailer.uni-marburg.de. † Philipps University. ‡ University of Nottingham. 1 Abbreviations: bPEI, (branched) polyethylenimine; lPEG, (linear) poly(ethylene glycol); mPEG, PEG-monomethyl ether; PLL, poly(L-lysine); MW, molecular weight; HMDI, hexamethylene diisocyanate; AFM, atomic force microscopy; N/P ratio, polycation-nitrogen/polyanion-phosphorous ratio; DLS, dynamic light scattering; PDI, polydispersity index; EtBr, ethidium bromide; LDH, lactate dehydrogenase.
12) most extensively with the result of reduced complement activation and an increase of DNA complex stability. However, PLL requires endosome disruptive reagents such as chloroquine (13) to attain sufficient transgene expression. As compared to PLL, PEI has successfully been used for gene delivery both under in vitro (14, 15) and in vivo (16, 17) conditions. PEI offers a high positive charge density and exhibits a strong proton buffer capacity over a broad pH range (18). The latter characteristic is advantageous for PEI since the proton-sponge effect (19) allows sufficient gene transfer without endosome disruptive reagents. PEGylated PEIs have been studied by several authors as potential gene delivery systems. For instance, PEI 25 kDa was grafted with three and 10 PEG 3400 Da blocks carrying a terminal galactose ligand for hepatocyte targeting (20). Kabanov and co-workers modified PEI 25 kDa with 37 and 61 PEG 5 kDa blocks and PEI 2 kDa with two blocks of PEG 8 kDa and one block of PEG 10 kDa (21, 22) to study the influence of PEG on oligonucleotide and plasmid DNA complexation. Instead, Choi et al. grafted PEI 25 kDa with 40 and 133 short blocks of PEG 550 Da (23) and applied this copolymer for in vitro gene transfer. While all of these groups grafted the PEG onto PEI first and formed the DNA complexes in a subsequent step, Wagner and co-workers first condensed
10.1021/bc025529v CCC: $22.00 © 2002 American Chemical Society Published on Web 06/22/2002
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DNA with PEI 800 kDa and subsequently grafted the complex with more than 1600 blocks of PEG 5 kDa (24, 25). Nevertheless, PEGylation of PEI has not been studied varying both the degree of substitution and the MW of PEG under similar experimental conditions to our knowledge. A retrospective comparison of literature data obtained with PEI of different MW and different experimental conditions is notoriously difficult. Therefore, our investigations were aimed at providing a PEI-g-PEG block copolymer sample set in which PEI is kept constant but the extent of PEGylation and the MW of the PEG is varied. Here, we use two series of PEI-g-PEG block copolymers in an effort to elucidate the influence of copolymer block structure on DNA condensation (copolymer structure/complex property relationship) and in a second step to compare the physicochemical properties of the complexes with their biological activity revealed in cell culture experiments (complex property/biological activity relationship). MATERIALS AND METHODS
Materials. bPEI with a MW of 25 kDa (Polymin water free, 99%) was a gift of BASF, Ludwigshafen, Germany. mPEG 550 Da and mPEG 5 kDa were obtained from Aldrich, Milwaukee, WI. Monoamino-mPEG (mPEGNH2) 20 kDa was purchased from Rapp Polymere, Tu¨bingen, Germany. All polymers were thoroughly dried in vacuo before use. HMDI (g99%) was from Fluka, Deisenhofen, Germany. Chloroform (Riedel-de Hae¨n, Seelze, Germany, g99%) was treated with HMDI for 4 h at 60 °C and distilled to remove any traces of water and ethanol. Diethyl ether (g99.5%) from Merck, Darmstadt, Germany, was distilled before use. Light petrol (g99%, 40-60 °C) was from Riedel-de Hae¨n. Three different types of DNA were used in this work. DNA from herring testes (Type XIV, 0.3-6.6 MDa, 40010 000 bp) and plasmid DNA pBR322 (2.9 MDa, 4363 bp) were from Sigma, Steinheim, Germany. Plasmid pGL3 control, luciferase reporter vector (3.5 MDa, 5256 bp) was from Promega, Heidelberg, Germany, and was amplified with a competent Escherichia coli strain JM 109 (Promega) according to a protocol from QIAGEN, Hilden, Germany (26). Block Copolymer Synthesis. Synthesis and characterization of the block copolymers has been described in detail elsewhere (27, 28). Briefly, PEG dissolved in anhydrous chloroform (c ) 200 g/L) was activated for the reaction with the amino groups of PEI with a 10-100fold excess of HMDI at 60 °C for 8-72 h depending on the MW of PEG (higher MW required longer reaction times). Unreacted HMDI was carefully removed by repetitive extraction with light petrol. Activated PEGs were reacted with PEI at concentrations of 10 g/L in anhydrous chloroform at 60 °C for 8-72 h. The reaction solutions were concentrated to 100 g/L by evaporation of the solvent and dropped into a 20-fold larger volume of diethyl ether to obtain the copolymer by precipitation. Finally, the products were dried in vacuo. Polymers were characterized by 1H and 13C nuclear magnetic resonance (NMR) spectroscopy, which verified the structure of the copolymers and allowed calculation of the composition of ethylenimine and ethylene glycol units in the copolymer. Size exclusion chromatography demonstrated the absence of unreacted PEG and PEI homopolymers. Thus, no further purification steps were necessary. Formation of the Copolymer-DNA Complexes. All complexes of DNA and polymer were prepared freshly
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before use. Polymer solutions were added to the DNA solutions in equal volumes, mixed by vortexing, and incubated for 10 min before use unless otherwise stated. DNA stock solutions were diluted to a concentration of 20 µg/mL with 10 mM NaCl at pH 7.4 (AFM experiments) or to 40 µg/mL (all other experiments) with 150 mM NaCl at pH 7.4. The polymer stock solutions were diluted with the same medium as above to the appropriate concentration depending on the required N/P ratio and filtered through 0.22 µm. DNA stock solutions were 2 mg/mL pGL3 plasmid in 10 mM TRIS HCl (pH 8) and 1 mM EDTA, 100 µg/mL pBR322 plasmid in pure reagent water, and 1.11 mg/mL herring testes DNA in 10 mM TRIS HCl (pH 7.4). Homopolymer and copolymer stock solutions were 0.5 mg PEI/mL (AFM experiments) and 0.9 mg PEI/mL (all other experiments) so that all polymer solutions contained the same PEI concentration. Physicochemical Characterization of CopolymerDNA Complexes. AFM. AFM images were conducted on a Dimension3000 Scanning Probe Microscope from Digital Instruments (Santa Barbara, CA) with a Nanoscope IIIa controller. The solution of polymer-DNA complex was prepared as described above and incubated for 5 min before it was deposited onto freshly cleaved mica and imaged under 10 mM NaCl solution. Immobilization was achieved using electrostatic forces between the opposite charges of the mica and polymerDNA complexes. All imaging was carried out in the tapping mode at a scan speed of approximately 2 Hz with 512 × 512 pixel data acquisition. V-shaped cantilevers with a pyramidal tip of silicon nitride were used (Park Scientific, CA). The experiments were repeated twice, and the observed structures were found to be reproducible. To determine the mean complex size from these images, the diameter at about half-height of 30 complexes was measured. DLS. Complex size measurements were carried out with a Zetasizer 3000 HS from Malvern Instruments, Worcs., U.K. at 25 °C (10 mW HeNe laser, 633 nm). Scattering light was detected at 90° angle through a 400 µm pinhole. Measurements yielding hydrodynamic diameters (Z average mean) and PDI were performed with count rates of about 100-400 kCps in the form of 10 runs of 60 min duration each and analyzed in the CONTIN mode. For data analysis, the viscosity (0.8905 mPas) and refractive index (1.333) of pure water at 25 °C were used. The instrument was calibrated with Nanosphere Size Standards (polymer microspheres in water, 220 ( 6 nm) from Duke Scientific, Palo Alto, CA. Laser Doppler Anemometry (LDA). The ζ-potential measurements of the complexes were carried out in the standard capillary electrophoresis cell of the Zetasizer 3000 HS from Malvern Instruments at position 17.0 and at 25 °C. Measurement duration was set to automatic. Average values of the ζ-potential were calculated with the data from eight runs. The instrument was calibrated with DTS5050 Electrophoresis Standards from Malvern Instruments. Agarose Gel Retardation Assay. Amounts of 40 µL aliquots of the complex solution were mixed with 4 µL of loading buffer [glycerol (85%) and TAE (40 mM Tris/HCl, 1% acetic acid, 1 mM EDTA, pH 7.4) in equal parts] and loaded onto an EtBr containing 1% agarose gel. Electrophoresis (Blue Marine 200, Serva, Heidelberg, Germany) was carried out at a voltage of 80 V (LKB Bromma 2197 Power Supply, Pharmacia, Freiburg, Germany) for 2 h in TAE running buffer solution. UV light (254 nm) detection was conducted with a Video Copy Processor P67E from Mitsubishi, Japan.
Polyethylenimine-graft-poly(ethylene glycol)
Biological Characterization of PEI-PEG-Copolymer-DNA Complexes. Hemolysis Assay. For hemolysis, fresh blood (700 g) from 6 month old rats (Fischer 344 rats, Institute for Pharmacology and Toxicology, University of Marburg) collected in heparinized tubes was centrifuged at 4 °C for 10 min and washed several times with phosphate-buffered saline (PBS) until the supernatant was colorless. A 500 µL amoount of a 2.5% (v/v) suspension of erythrocytes was mixed with 500 µL of complex solution in Eppendorf cups. After it was incubated for 2 and 60 min at 37 °C in a shaking water bath, the blood cells were removed by centrifugation and the supernatant was investigated spectroscopically at 540 nm for the release of hemoglobin. Experiments were performed in triplicate. As controls, PBS (negative ) 0%) and 0.2% Triton X-100 solution (positive ) 100%) were used. Aggregation of Erythrocytes. To study polymer/DNA complex-induced aggregation of erythrocytes, rat blood (see hemolysis test) was applied. Ringer’s solution (with the addition of sodium citrate, pH 7.4) was used to prevent coagulation. The blood was washed several times with Ringer’s solution until the supernatant was colorless as described above. Finally, the erythrocytes were diluted with Ringer’s solution 1:50. A 200 µL amount of this cell suspension was treated with 100 µL of polymer solution in 24 well plates (Nunc). After it was incubated for 2 h at 37 °C, pictures were taken of the cells with a Contax RTS 2 camera from AGFA (ASA 100) fitted to a reverse phase contrast microscopy (Nikon TMS) at 40 times magnification. Experiments were performed in triplicate. LDH Assay. In vitro cytotoxicity of the complexes with herring testes DNA was evaluated using an LDH assay with 3T3 mouse fibroblasts (500 000 cells/well, 4 h incubation). As reference, PBS (negative, 0%) and 0.1% (w/w) Triton X-100 solution (ICN, Eschwege, Germany) in PBS (positive, 100%) were applied. The LDH content was measured photometrically (340 nm) using a commercial test kit (DQ 1340-K, Sigma) as described in more detail earlier (28). Experiments were performed in triplicate. Transfection Experiments. Transfection activity of the copolymer complexes was studied on 3T3 mouse fibroblasts (NIH 3T3, Swiss mouse embryo, ATCC, Rockville, Maryland) using a Luciferase assay kit from Promega. Measurements of relative light units (RLU) were conducted on a Sirius Luminometer from Berthold, Pforzheim, Germany. Protein quantification was performed with freshly prepared bicinchonic acid (BCA) reagent (Pierce, Rockford, IL) and was carried out with an enzyme-linked immunosorbent assay (ELISA) plate reader from Dynatech MR 5000, Denkendorf, Germany, at 570 nm. Experiments were performed in triplicate as described in more detail in ref 30. RESULTS AND DISCUSSION
Two series of block copolymers were prepared by grafting lPEG onto bPEI using a straightforward synthesis route (Scheme 1). Diisocyanate HMDI was used as a linker generating hydrolytically stable urethane bonds between PEG and linker and urea bonds between PEI and linker. In the first series (Scheme 2, horizontal), the PEI (25 kDa) was grafted with different degrees of substitution using PEG blocks all of MW 5 kDa in order to study the quantitative effect of PEG blocks. As a result, the polycationic domain of the copolymers was increasingly surrounded by PEG blocks in this series. The second
Bioconjugate Chem., Vol. 13, No. 4, 2002 847 Scheme 1. Copolymersa
Synthesis
of
bPEI(25k)-g-lPEG
a The PEG has a linear structure, and the PEI has a hyperbranched structure.
series (Scheme 2, vertical) was arranged with copolymers all containing an equal weight percent of PEG and PEI (about 50:50%) but with PEGs of different molecular weights (MW 550-20 000 Da). Thus, we varied the copolymer structure in such a way that on one extreme PEG is homogeneously distributed in short blocks within the PEI domain, whereas in the other extreme a clear separation of the PEG and the PEI domain is realized. In other words, the copolymer structure is varied from multiarm grafting to a diblock structure. For these graft (g) copolymers, we chose following nomenclature: bPEI(25k)-g-lPEG(x)n where b and l denote a branched or linear structure. The numbers in brackets (25k or x, where x ) 550, 5k, or 20k) represent the MW of the PEI or the PEG block and the index n is the average number of PEG blocks per one PEI macromolecule. The index n was calculated from integration of the 1H NMR signals with the MW values of the homopolymers given by the suppliers. Size and Morphology of the Complexes. To study the influence of copolymer structure on size and shape of the complexes formed with plasmid DNA, we performed AFM experiments in aqueous solution. While transmission electron microscopy (TEM) has been frequently used to characterize DNA/polycation complexes (20-22), the advantage of AFM stems from the direct observation of these complexes in aqueous solution. Samples for TEM are visualized in vacuo and need to be pretreated and stained, e.g., with zinc uranyl acetate. Therefore, dimensions and structure of DNA/polycation complexes determined in aqueous media using AFM and by TEM could be different. In this study, physiological salt concentrations (150 mM NaCl) as used for the biological experiments were not feasible technically due to formation of salt crystals during the AFM imaging process. Therefore, a low salt concentration of 10 mM NaCl and pH 7.4 was selected. Because immobilization was based on electrostatic forces between the negative charge of mica substrate and the complex, the latter had to exhibit a distinct positive net charge. Therefore, we chose the N/P ratio 9 for this study (compare with ζ-potential measurements below). The homopolymer PEI showed images of spherical and compact complexes of 142 ( 59 nm diameter (Figure 1A and Figure 2). The first copolymer series (images A-D)
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Scheme 2. Schematic of the bPEI(25k)-g-lPEG Copolymer Sample Seta
a The black part of the structure represents the cationic branched PEI, and the gray part represents the nonionic linear PEG blocks. It should be remarked that the structures are reduced to two dimensions. Thus, the density of the PEG shield in (3D) reality is smaller than it appears in this scheme. The copolymer bPEI(25k)-g-lPEG(550)35 has two possible structures; however, PEG is more likely homogenously distributed within the PEI than concentrated at the outer sphere of the PEI (core-shell structure).
Figure 1. AFM images of polymer/plasmid DNA complexes at N/P ratio 9. Image A shows PEI 25 kDa homopolymer DNA condensates. Images B-D show DNA-polymer condensates produced with plasmid DNA and bPEI(25k)-g-lPEG(5k)n where n ) 2, 6, and 15, respectively. Images E and F show DNA condensates produced with bPEI(25k)-g-lPEG(550)35 and PEI(25k)-g-PEG(20k)1, respectively. Scale bars are equivalent to 250 nm. The color-encoded height scale extends 15 nm.
demonstrated that PEG 5 kDa significantly reduced the diameter of the complexes from 142 nm to about 60 nm, but with an increasing number of PEG blocks, DNA condensation was affected and complexes lost their spherical shape. Whereas the copolymer containing 2 PEG blocks gave spherical complexes with diameters of 77 ( 36 nm, bPEI(25k)-g-lPEG(5k)6 showed a mixture of spherical and rodlike particles (diameter 61 ( 28 nm). When PEI was grafted with 15 PEG blocks, complexes of ill-defined shape were found (diameter 60 ( 46 nm).
Figure 2. DNA-polymer complex size as determined by AFM (n ) 30) and DLS (n ) 10) at N/P ratio 9 in 10 mM NaCl. The error bars indicate the standard deviation (SD). In the case of the AFM results, the SD reflects the size distribution.
Furthermore, some of the complexes exhibited stringy structures, which can be attributed to poorly condensed DNA. Complex size was also determined by DLS in the same medium as used for AFM (10 mM NaCl, pH 7.4). Hydrodynamic diameters are shown in Figure 2 in comparison with the results of the AFM experiments. Differences in complex size were much smaller as compared to the AFM results, and no specific trend could be
Polyethylenimine-graft-poly(ethylene glycol)
Figure 3. Hydrodynamic diameters of complexes assembled by plasmid DNA with bPEI 25 kDa and bPEI-g-lPEG copolymers at different ionic strength and different N/P ratios as determined by DLS.
observed for the first series. The trend found by AFM that PEG 5 kDa reduces the size of the complex was observed by DLS only for bPEI(25k)-g-lPEG(5k)2. Their complexes (88.7 ( 2.2 nm) were on average slightly smaller than complexes formed by the homopolymer PEI (94.5 ( 1.1 nm). However, copolymers with 6 and 15 PEG blocks formed complexes with slightly larger average diameters of about 105 nm probably due to the rodlike shape of some of the complexes as observed in AFM. In the second series, another trend is observed in AFM (Figure 1C,E,F): Complexes gradually lose their compact and spherical shape with decreasing MW of PEG. Copolymers with one long PEG 20 kDa formed small (51 ( 23 nm) and clearly spherical complexes, whereas copolymers with many short PEG 550 Da gave rather large complexes (131 ( 60 nm), which were fluffy or diffuse and almost shapeless. For this series, DLS measurements revealed the same trend: For bPEI(25k)-g-lPEG(20k)1, a small complex diameter of 68.7 ( 4.3 nm was found, whereas in the case of bPEI(25k)-g-lPEG(550)35 larger diameters (110.4 ( 12.0 nm) were determined. The results of both the AFM experiments and the DLS measurements demonstrated that DNA complexation with PEI-g-PEG is strongly dependent on the block copolymer structure. In the case of PLL-g-PEG copolymers, a negative influence of PEG and other hydrophilic polymers on the condensation of DNA was observed (11, 31). Similar disadvantageous effects were anticipated for PEI; therefore PEGylation of the PEI/DNA complex was carried out as a second step after condensation (25). This procedure may, however, be impractical in a clinical setting. Here, we demonstrated that PEG does not necessarily interfere with the condensation process of DNA. Attaching only one or two PEG blocks to PEI can even enhance DNA condensation forming compact and spherical complexes with smaller sizes as compared to homopolymer PEI complexes. Using DLS, complexes were also studied at high ionic strength (150 mM NaCl, pH 7.4) and at different N/P ratios (Figure 3). Under these (more physiological) conditions, all polymers formed larger complexes as compared to low salt concentration. However, with an increasing N/P ratio, the complex size was reduced again. Two exceptional polymers were found. At a low N/P ratio (4.5),
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Figure 4. Stabilization study of the complexes assembled by plasmid DNA with bPEI 25 kDa and bPEI-g-lPEG at N/P 4.5 and 150 mM NaCl. Complex size (diameter) is as a function of incubation time as determined by DLS.
Figure 5. ζ-Potential of plasmid DNA complexes with PEI 25 kDa and bPEI-g-lPEG block copolymers at different ionic strength and at different N/P ratios as determined by laser Doppler anemometry.
PEI and bPEI(25k)-g-lPEG(550)35 did not give stable complexes. Over a period of 30 min, complex size increased from 200 to 600 nm and from 270 to 900 nm, respectively (Figure 4). In contrast, all copolymers with PEG of 5 and 20 kDa formed stable complexes at N/P 4.5 (copolymers bPEI(25k)-g-lPEG(5k)n where n is 2 or 15 are not shown in Figure 4). A stabilizing effect of PEG against aggregation of PEI/DNA complexes has been noted previously (21, 25). Short PEG blocks seem to provide little stabilization, and a MW of PEG g 5 KDa is necessary to obtain this effect. At higher N/P ratios (g9), all homo- and copolymers formed stable complexes, which are probably stabilized electrostatically under these conditions by a larger excess of polycations present. Surface Charge of the Complexes. To estimate the surface charge of the complexes, we measured the ζ-potential of the complexes by LDA. The results are shown in Figure 5. In the first series of bPEI(25k)-glPEG(5k)n copolymers, the ζ-potential is reduced with an
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increasing number n of PEG blocks. This is true for different salt concentrations and different N/P ratios. Thus, PEG 5 kDa blocks seem to orientate toward the surface of the complex and shield its core with the cationic net charge. In the case of the copolymers, the ζ-potential increases with increasing N/P ratios. The ζ-potential did not increase further from N/P 20 to 50 since free copolymer was present at N/P 50. In the case of homopolymer PEI, even at low N/P ratio 4.5, ζ-potential is highly positive and is found in the range of +25 to +35 mV. Similar values have been reported by others (24, 25, 32). A ζ-potential of +35 mV seems to be the maximum surface charge of the PEI-DNA complexes. PEI was reported to possess a ζ-potential of +37 mV (33). In the second series with the copolymers containing about 50% PEG, the ζ-potential is reduced with increasing MW of PEG. Therefore, PEG 5 kDa and especially PEG 20 kDa are able to shield the charged core of the complex. The high ζ-potential of complexes formed with bPEI(25k)-g-lPEG(550)35 points to the poor shielding properties. As we have previously shown using differential scanning calorimetry (28), bPEI(25k)-g-lPEG(550)35 probably did not form a simple core-shell structure in which the PEG blocks surrounded the positively charged PEI core. During the synthesis, the short PEG blocks might penetrate into the branched PEI structure and react also with the inner amino groups. This would yield a copolymer where PEG is more homogeneously distributed over the PEI macromolecule as schematically shown in Scheme 2. This structure could explain the high ζ-potential of the complex. DNA Complexation and Condensation. Finally, to study DNA complexation and condensation as a function of N/P ratio, agarose gel retardation assays were performed. The gels are shown in Figure 6. No difference was found for the copolymers from the first series with PEG 5kDa: Complete retardation of DNA (indicating complete DNA complexation) was achieved at N/P ) 2.02.2 whereas for complete exclusion of EtBr (indicating complete DNA condensation) a further excess of the polycation was needed (N/P ) 4.5, data not shown). In the case of homopolymer PEI, DNA was complexed completely at N/P ) 1.6 and condensed at 3.0. Thus, grafting PEI with PEG 5 kDa slightly hindered the interaction with DNA. When PEI was grafted with short PEG blocks as in the case of copolymer bPEI(25k)-glPEG(550)35, the complexation was hindered slightly (N/P ) 2.0) but condensation was realized at the same N/P ratio (N/P ) 3.0). Here again, we found that short PEG blocks behaved differently than longer PEG chains: PEG 550 Da did not stabilize the complex against aggregation, it did not reduce the positive surface charge, and here, it did not affect DNA condensation. However, the most interesting result from the agarose gel retardation assay is the enhanced DNA condensation observed for copolymer bPEI(25k)-g-lPEG(20k)1 already at N/P ) 2.6. This is in line with the results from AFM images where small and compact particles were found. The excellent DNA condensation might be attributed to the special copolymer structure. With only one PEG block on average, this is a diblocklike copolymer and there might be a PEI domain and a PEG domain clearly separated from each other. Thus, the DNA seems to interact with the PEI domain without interference from PEG. Furthermore, some authors reported the DNA condensation ability of PEG (34, 35). Therefore, PEG might additionally participate in the condensation process in a synergistic manner. Hemocompatibility of the Complexes. The blood compatibility of PEI-g-PEG/DNA complexes was screened
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Figure 6. Agarose gel electrophoresis of the complexes of plasmid DNA with bPEI 25 kDa and bPEI-g-lPEG block copolymers. Lane 1, N/P ) 0 (plasmid only); lanes 2-14, N/P 0.6-3.0 with increments of 0.2. N/P ratios of completed DNA retardation (left mark) and of completed ethidium bromide exclusion (right mark) sign the appropriate lanes. N/P ratio 4.5 was determined with another gel experiment (data not shown).
using a hemolysis and an aggregation of erythrocytes under in vitro conditions. These assays are a prerequisite for the intravenous administration of PEI-g-PEG/DNA complexes in animals or humans. The results of the hemolysis assay are presented in Table 1. In a short term experiment (incubation time with erythrocytes, 2 min; data not shown), the acute phase of an intravenous administration is reflected. No hemolysis was observed after short incubation time for any of the PEI-g-PEG preparations. Complexes with PEI 25 kDa showed a slight hemolytic activity (1-2%). The long term experiment (60 min incubation) still showed only negligible hemolysis for the complexes of PEI and most copolymers. PEI, bPEI(25k)-g-lPEG(5k)2, and bPEI(25k)g-lPEG(550)35 caused more hemolysis (up to 5%) after 60 min in comparison with the results after 2 min of incubation. Only bPEI(25k)-g-lPEG(550)35 exhibited a remarkable value of 12% at very high N/P ratios, which is still tolerable since substances are classified as nonhemolytic when hemolysis remains
